Enhancing durability and sustainable preservation of Egyptian stone monuments using metabolites produced by Streptomyces exfoliatus

Despite their threatens for Egyptian stone monuments, A few studies focused on using biocontrol agents against deteriorative fungi and bacteria instead of using chemical assays that leave residuals leading to human toxicity and environmental pollution. This work aims to isolate and identify fungal and bacterial isolates that showed deteriorative activities from stone monuments in Temple of Hathor, Luxor, Egypt, as well as determine the inhibitory activity of metabolites produced by Streptomyces exfoliatus SAMAH 2021 against the identified deteriorative fungal and bacterial strains. Moreover, studying the spectral analysis, toxicological assessment of metabolites produced by S. exfoliatus SAMAH 2021 against health human cell fibroblast, and colorimetric measurements on the selected stone monuments. Ten samples were collected from Temple of Hathor, Luxor, Egypt. Three fungal isolates and one bacterial isolate were obtained and identified as A. niger isolate Hathor 2, C. fioriniae strain Hathor 3, P. chrysogenum strain HATHOR 1, and L. sphaericus strain Hathor 4, respectively. Inhibitory potential of the metabolites in all concentrations used (100–25%) against the recommended antibiotics (Tetracycline 10 µg/ml and Doxycycline (30 µg/ml) showed an inhibitory effect toward all tested deteriorative pathogens with a minimum inhibition concentration (MIC) of 25%. Cytotoxicity test confirmed that microbial filtrate as the antimicrobial agent was safe for healthy human skin fibroblast with IC50 of < 100% and cell viability of 97%. Gas chromatography analysis recorded the existence of thirteen antimicrobial agents, Cis-vaccenic acid; 1,2-Benzenedicarboxylic acid; ç-Butyl-ç-butyrolactone and other compounds. Colorimetric measurements confirmed no color or surface change for the limestone-treated pieces. The use of the metabolite of microbial species antimicrobial as a biocontrol agent raises contemporary issues concerning the bio-protection of the Egyptian monuments to reduce chemical formulas that are toxic to humans and pollute the environment. Such serious problems need further investigation for all kinds of monuments.


Isolation of the deteriorative fungi and bacteria. Poured plate technique was recommended for
counting the selected microbes. For the isolation of fungal and bacterial isolates, OGYA and PCA media were prepared and placed into plates, respectively. All plates were inoculated and incubated for 72 h at 25 °C for fungi and 24 h at 30 °C for bacteria. All colonies were collected and purified using OGYA and PCA subcultures for fungal and bacterial species, respectively, and then stored at 4 °C for further investigations 16 . Standard inoculum. Standard inoculum of (1.6-3.8 × 10 8  www.nature.com/scientificreports/ saline solution yielded spore suspensions for fungal isolates. The spore suspensions (1.1 × 10 8 /mL) collected were utilized as standard inoculum in shake flask tests. Scratched slant cultures of S. exfoliatus SAMAH 2021 were resuspended in 50 mL of sterile saline water, and spore concentration was adjusted to 16 × 10 9 spore/mL 17 .

Identification of deteriorative bacterial isolates. Phenotypic identification.
Isolates were identified based on their cultural and cell morphological features. Bacterial isolates were stained using Gram and spore staining. Fungal isolates were grown on slide cultures.
Bacterial genotypic identification. Bacterial DNA was extracted, and gene sequencing was applied using molecular based approach using polymerase chain reaction (PCR) to partially amplify the 16S rRNA gene sequence using the two universal primers (F1: 5′ AGA GTT T (G/C) ATC CTG GCT CAG 3′ and R1 5′ ACGG (A/C) TAC CTT GTT ACG ACTT 3′). The partially amplified PCR product was purified using a QIA quick gel extraction kit (Qiagen, Germany). Macrogen company (South Korea) sequenced the 16S rRNA of the purified PCR product. Using BioEdit version 7.0.4, sequence readings were clipped and aggregated, and ClusterW version 4.5.1 was used to align the resulting genomic information. The NCBI database was employed to conduct BLAST inquiries 18 . MEGA 11 software was employed to build phylogenetic trees that use the neighbor-joining cladogram 19 -TCC GTA GGT GAA CCT GCG G-3′), and ITS4: (5′-TCC TCC GCT TAT TGA TAT GC-3′). The ddNTPs were incorporated into the reaction mixture and the corresponding primers for sequencing the purified recombinant result. Biological antimicrobial activity of S. exfoliates metabolites. The produced standard S. exfoliatus SAMAH 2021 inoculum was inoculated into CSB medium at 5% v/v (2 × 10 7 spores/mL) and incubated at 30 °C for 240 h at 120 rpm. Ten milliliters were taken at 10d intervals. For 15 min, cultures were centrifuged at 10,000 rpm. Pellets were collected to determine cell dry weight (CDW), and the supernatant was collected to determine antibacterial activity as an inhibition zone diameter (IZD) in centimeters (cm). All experiments were done in triplicate. The logarithmic phase regression coefficient was estimated based on the correlation between time (h) and IZD (cm). According to Maier et al. 20 , the specific inhibition rate (d) was calculated as follows: where X = IZD after the time (t) and X 0 = IZD at the beginning time (t 0 ).
(1) Specific inhibition rate(µ d /h) = (ln X−ln X 0 ) / (t − t 0 )  The CIE L* a* b* system was used to record the color variations, with the L value equating to brightness, the "a" value to red-green, and the "b" value to yellow-blue. The total color changes (E) before and after treatment were calculated using the following Equation 24 : where L (lightness), a (red/green axis), and b (yellow/blue axis) values were recorded.
Gas chromatography (GC/MS) analysis for S. exfoliatus SAMAH 2021 metabolites. Extracted products of S. exfoliatus SAMAH 2021 were dried over anhydrous Na 2 SO 4 using a rotary evaporator, then dissolved by methanol. A capillary column TG-5MS (30 m × 0.25 mm × 0.25 m film thickness) and a Trace GC-TSQ mass spectrometer (Thermo Scientific, Austin, TX, USA) were used. The column temperature was maintained at 50 °C and raised by 5 °C/min until reaching 250 °C, then maintained for 2 min. expanded by 30 °C/min to an ultimate temperature of 300 °C and held for 2 min. Helium was employed as the carrier gas, with a constant flow rate of 1 mL/min, and temperatures of the injector and MS transfer line were maintained at 270 and 260 °C, respectively. The auto sampler AS1300 paired with the GC in split mode automatically injected diluted samples of 1 μL with a solvent delay of 4 min. EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 50-650 in full scan mode. The ion source temperature was set at 200 °C. The components were identified by comparison of their mass spectra with those of WILEY 09 and NIST 14 mass spectral database 25 .

Cytotoxicity of S. exfoliatus SAMAH 2021 metabolites. Human skin fibroblast (HSF) cells were
obtained from Nawah Scientific Inc. (Mokatam, Cairo, Egypt). DMEM media with added antibiotics (streptomycin 100 mg/mL, penicillin 100U/mL, and heat-inactivated fetal bovine serum 10%) were prepared in a CO 2 humid atmosphere of 5% humidity (v/v) at 37 °C. SRB assay was carried out for the determination of cell viability 26 .
Statistical analysis. All samples and collected data were statistically analyzed using IBM® SPSS® Statistics software (2017). A Tukey test at a P-value of 0.05 was applied.

Results
Isolation of the deteriorative microflora. Ten biofilm samples were taken from the degraded bas-relief surfaces of the Hathor temple and inoculated on PCA and OGYA plates for microbial screening. Four isolates were obtained (3 fungal, F1, F2, F3, and one bacterial, B1, isolates). Following successive purification processes, all isolates were identified phenotypically and genotypically.
Identification of the deteriorative isolates. Phenotypic identification. The bacterial isolate, B1, had a round colony with smooth surface end edges and off-white color. Microscopic examinations confirmed its shape and motility as it had a long rod shape, motile and terminal spores. Fungal isolate F1 had black conidial spores, while F2 isolate exhibited greenish colonies with branching or simple conidiophores in brush-like clusters. Isolate F3 had orange-salmon conidial hypha and dark melanized structures, as shown in Fig. 2.     (Fig. 4). The correlation coefficient of CDW and IZD of S. exfoliatus SAMAH 2021 metabolites against identified deteriorative strains reflected a strong correlation ranging between 0.93 and 0.99% for all tested strains (Fig. 5). Growth kinetics for the logarithmic phase revealed that the specific inhibition rate (μ d ) was 0.05, 0.04, 0.03, and 0.021 cm.h -1 for the stone monuments deteriorative strain of P. chrysogenum, A. niger, C. fioriniae, and L. sphaericus, respectively, as presented in (Fig. 6).
Antibiotic and antimicrobial sensitivity for the four obtained deteriorative strains. Table 1 showed that antimicrobial compounds had strong antimicrobial efficacy against pathogenic G +ve and G −ve bacteria, fungi, and yeast when compared to the most widely used commercial antibiotics (tetracycline and doxycycline). At 100% concentration, antimicrobial compounds had an IZD ranging from 2.1 to 5.2 cm, while the commercial antibiotic had an IZD ranging from 0.85 to 4.17 cm. At concentrations ranging from 25 to 75%,     Figure 6. Inhibition rate (cmh −1 ) for the stone monuments deteriorative strains during the logarithmic phase of S. exfoliatus SAMAH 2021 antimicrobial.    Table 3 and Fig. 10.

Cytotoxicity.
Using an MTT assay, four different concentrations of the active metabolites from S. exfoliatus SAMAH 2021 (0-100%) were examined towards standard HSF cells. Figure 11 showed no adverse consequences at all doses of the antimicrobial products in functional HSF cells. Cell viability increased to 100%, where it was 97%. 100% of HSF cells were viable under the control condition. We calculated the half-maximal inhibitory concentration (IC50), which was > 100%, using GraphPad Prism (5). When compared to the control treatment, microscopic pictures for S. exfoliatus SAMAH 2021 antimicrobial agents exhibited no cytotoxicity.

Discussion
The microbial deterioration of Egyptian stone monuments emphasizes the need for finding efficient solutions to mitigate the effects of biodeterioration and enhance the international consciousness about the biopreservation of our historical heritage. Studies should focus on discovering new alternatives for the chemical antimicrobial compounds which had harmful effects on man and environment as well 27 . Our results revealed that 75% of the collected deteriorative isolates were fungi rather than bacteria indicating the gross contamination of museums, temples, and tombs. This fungal contamination is due to the low temperatures and high relative humidity to reach 70% in indoors that encourages fungal growth by germinating and spreading their spores especially xerophilic and xerotolerant species such as Aspergillus sp., Penecillium sp., and Wallemia sp. 28 . Dessimentaion of fungal strains in museums is also influenced by the presence of air borne minerals, carbonates, and other combounds 29 . There are two types of deteriorative fungi, the Epileptic fungi (living on the rock) and endolithic fungi (living inside pores and fissures) 30 such as such as Penicillium frequentens, and Aspergillus fumigatus. Black fungi (Aspergillus sp.) is responsible for stone monuments biodeterioration. Likewise, it has  www.nature.com/scientificreports/ been found that Penicillium sp. was related to the formation of dark biofilms on the outer layer of landmarks 31 . L. sphaericus is a harmful bacterium forms reistant endospores to high temperatures, synthetic compounds, and intense light causing biofilm formation on stone surfaces 32 . Our findings were consistent with Salvadori and Municchia 33 , who recovered A. niger and Penicillium sp. from stone monuments. Also, P. frequentens and Cladosporium cladosporoides were isolated by Mohammadi et al. 34 from stone monuments. It was also reported that A. niger was the most abundant species found on limestone, marble, and sandstone. In addition to previous, A. flavus and Fusarium roseum were found on land sites, limestone, marble, and sandstone 35 . Streptomyces sp., is well known as the greatest producer for secondary metabolites especially antimicrobial products 36 . In the current study, numerous bioactive substances that act as antimicrobial agents were identified by (GC-MS) analysis. Ethyl iso-allocholate; 13-heptadecyn-1-ol and 9,12,15-Octadecatrienoic acid 2,3-dihydroxy propyl ester, (Z, Z, Z) possess antimicrobial activity (antiviral, anti-bacterial, and anti-fungal) 37 . The primary metabolites of S. exfoliatus SAMAH 2021 are fatty acids and fatty acid ester. Generally, they were more abundant in S. exfoliatus SAMAH 2021 growth cultures such as Cis-Vaccenic acid; 9-Octadecenoic acid (Z)-,2-hydroxy-1-(hydroxymethyl) ethyl ester and Oxiraneoctanoic acid, 3-octyl-, methyl ester.
Streptomyces has reported to produce peptides/glycopeptides, angucyclinone, tetracyclines, phenazine, macrolide, anthraquinone, polyene, nonpolyene, benzoxazolophenanthridine, heptadecaglycoside, lactones, and other antibiotics 38 39 . It is well known that unsaturated fatty acids have high antimicrobial activity rather saturated fats due to the presence of double bonds 40 . Moreover, free fatty acid surfactants have a high potential to damage cell membranes' stability as well as the inhibition of electron transport chain-related enzymes 41 . Tetradecan-1-OL is one of the found bioactive compounds. It likes fatty alcohol and could be used for antibacterial and antifungal purposes. Also, 1, 2-benzene dicarboxylic acid has antibacterial properties. Chemically, ç-Butyl-ç-butyrolactone and 5-Hydroxymethyldihydrofuran-2-one are described as intramolecular esters of hydroxycarboxylic acids with variable cyclic diameters 42 . Lactones are cytotoxic, antiviral, and antibacterial agents that inhibit microbial growth 43 .
The color variations of experimental sandstone samples before and after treatment with S. exfoliatus SAMAH 2021 metabolites indicate the reflection spectra of materials damaged by the obtained strains of C. fioriniae strain Hathor 3, P. chrysogenum strain HATHOR 1, and L. sphaericus strain Hathor 4. The color difference (L) values, the rate of the darkness(%) and color difference (ΔE) for the deteriorating stone samples revealing that the limestone didn't affect by S. exfoliatus SAMAH 2021 metabolites.
Our results emphasized that metabolites produced by S. exfoliatus SAMAH 2021 are safe to human and could be used as a biocontrol agent against the most deteriorative fungi and bacteria for Egyptian stone monuments. However, more further studies are indeed needed to ensure the safety concerns of metabolite's treatment on the monuments along time.

Conclusion
In the current study, three deteriorative fungi and one bacterial strain (A. niger, C. fioriniae, P. chrysogenum, and L. sphaericus) that showed deteriorative activity on the Egyptian stone monuments were picked up and isolated from the Temple of Hathor. Applying S. exfoliatus SAMAH 2021 antimicrobial products was an effective method for protecting and preserving our Egyptian stone monuments as it had an inhibitory effect toward all tested deteriorative pathogens with a minimum inhibition concentration (MIC) of 25%. GC-MS analysis showed the existence of 13 compounds in the S. exfoliatus SAMAH 2021 products. The light change studies indicated no color or surface change for the limestone piece treated with S. exfoliatus SAMAH 2021 antimicrobial products as well as the safe usage of such antimicrobial products as proved by the toxicity experiment.